DOCSLIB.ORG

Development of Sirna Payloads to Target KRAS -Mutant Cancer

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Development of Sirna Payloads to Target KRAS -Mutant Cancer Published OnlineFirst August 6, 2014; DOI: 10.1158/2159-8290.CD-13-0900 RESEARCH ARTICLE Development of siRNA Payloads to Target KRAS -Mutant Cancer Tina L. Yuan 1 , Christof Fellmann 2 , Chih-Shia Lee 3 , Cayde D. Ritchie 1 , Vishal Thapar 2,4 , Liam C. Lee 3 , Dennis J. Hsu 3 , Danielle Grace 2,4 , Joseph O. Carver 3 , Johannes Zuber 2,5 , Ji Luo 3 , Frank McCormick 1 , and Scott W. Lowe 2,4,6 Downloaded from cancerdiscovery.aacrjournals.org on October 4, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 6, 2014; DOI: 10.1158/2159-8290.CD-13-0900 ABSTRACT RNAi is a powerful tool for target identifi cation and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must com- bine potent siRNA payloads with reliable in vivo delivery for effi cient target inhibition. We used a func- tional “Sensor” assay to establish a library of potent siRNAs against RAS pathway genes and to show that they effi ciently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/CRAF) as promising strategies to treat KRAS -mutant colorectal cancer. We further demonstrate that improved therapeutic effi cacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS + PIK3CA/B ). The customizable nature of Sensor siRNA payloads offers a universal platform for the combination target identifi cation and development of RNAi therapeutics. SIGNIFICANCE: To advance RNAi therapy for KRAS -mutant cancer, we developed a validated siRNA library against RAS pathway genes that enables combination gene silencing. Using an in vivo model for real-time siRNA delivery tracking, we show that siRNA-mediated inhibition of KRAS as well as RAF or PI3K combinations can impair KRAS -mutant colorectal cancer in xenograft models. Cancer Discov; 4(10); 1182–97. ©2014 AACR. INTRODUCTION RNAi provides an alternative therapeutic approach to small- molecule and antibody-based therapeutics for inhibiting gene Mutations in the RAS family of small GTPases, particularly function. RNAi can, in principle, be applied to reversibly silence KRAS, occur in 30% of all human cancers and are often asso- any target gene (reviewed in refs. 8 and 9 ), thereby increasing ciated with resistance to chemotherapy and targeted therapy. the druggable landscape from 10% to virtually 100% of the Somatic mutations lock RAS in the GTP-bound state, leading genome ( 10, 11 ). Because all siRNAs bear structural similarity to constitutive activation of its downstream effector path- and are likely to have comparable pharmacokinetic profi les, ways. Despite intense efforts, pharmacologic inhibition of their use as therapeutics would facilitate drug formulation, KRAS itself ( 1 ) and inhibition of individual effector kinases preclinical testing, and development of combination therapies. downstream of KRAS such as RAF ( 2, 3 ) and MEK ( 4–6 ) have Although delivery is still a major challenge, more reliable lipid so far been unsuccessful in treating KRAS -mutant tumors. and polymeric nanoparticles are in development to deliver Combinations of MEK and PI3K inhibitors are currently siRNA payloads to target tissues (reviewed in ref. 12 ). Regard- being evaluated in clinical trials, although toxicity in normal less, treatment effi cacy would benefi t from the development tissue could limit their therapeutic window ( 7 ). Thus, effec- of optimized siRNA payloads that potently and specifi cally tive and targeted treatment against KRAS-driven cancer is an silence well-validated target genes at low dose. urgent and unmet clinical need. Beyond its potential as a therapeutic modality, RNAi is a useful tool for identifying and validating new drug targets. This has proved particularly powerful in cancer research, where 1 Helen Diller Family Comprehensive Cancer Center, University of Cali- fornia, San Francisco, San Francisco, California. 2 Cold Spring Harbor shRNA or siRNA screens have been used to identify genes that Laboratory, Cold Spring Harbor, New York. 3 Laboratory of Cancer Biol- are selectively required for the proliferation and survival of ogy and Genetics, Center for Cancer Research, National Cancer Institute, cancer cells. However, the identifi cation and current in silico 4 Bethesda, Maryland. Memorial Sloan Kettering Cancer Center, New York, prediction of effective shRNAs and siRNAs remains imprecise, New York. 5 Research Institute of Molecular Pathology, Vienna, Austria. 6 Howard Hughes Medical Institute, New York, New York. resulting in low success rates. Consequently, screening high- order shRNA/siRNA combinations is not possible without Note: Supplementary data for this article are available at Cancer Discovery fi rst establishing a collection of functionally validated RNAi Online (http://cancerdiscovery.aacrjournals.org/). triggers. In addition, off-target effects, which can be due to T.L. Yuan, C. Fellmann, and C.-S. Lee contributed equally to this article. sequence-dependent and sequence-independent gene deregu- Current address for C. Fellmann: Mirimus, Inc., Cold Spring Harbor, New lation (reviewed in ref. 13 ), must be minimized for meaningful York. interpretation of phenotypic outcomes. Corresponding Authors: Scott W. Lowe, Memorial Sloan Kettering Cancer To overcome these limitations, we used a previously Center, 415 East 68th Street, Z-1114, New York, NY 10065. Phone: 646- described “Sensor” assay ( 14 ) to generate a functionally 888-3342; Fax: 646-888-3347; E-mail: [email protected] ; Frank McCor- validated library of RNAi sequences against RAS path- mick, [email protected] ; and Ji Luo, [email protected] . way genes. We show that Sensor siRNAs effi ciently ablate doi: 10.1158/2159-8290.CD-13-0900 their gene targets at low nanomolar concentrations in vitro , © 2014 American Association for Cancer Research. which decreased off-target effects and enabled the use of OCTOBER 2014CANCER DISCOVERY | 1183 Downloaded from cancerdiscovery.aacrjournals.org on October 4, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 6, 2014; DOI: 10.1158/2159-8290.CD-13-0900 RESEARCH ARTICLE Yuan et al. high-order siRNA combinations to codeplete multiple correlation between the initial algorithmic rank and the rank genes. By applying Sensor siRNAs in vitro and in vivo , we obtained through the biologic Sensor assay ( Fig. 1E ), show- identifi ed single-gene and combination-gene payloads that ing that prediction tools alone are still insuffi cient to select inhibit the growth of KRAS -mutant colorectal cancer. Thus, the most potent shRNAs (see also Supplementary Fig. S1C the use of Sensor siRNAs for target discovery and develop- and S1D). Conversely, the Sensor assay identifi ed potent ment of customized siRNA payloads can lead to nanoparticle- shRNAs for nearly every gene, with top-ranked shRNAs show- based treatments for KRAS -mutant cancer and provide a ing similar scores across all genes ( Fig. 1F and Supplementary blueprint for similar strategies to target other key nodes in Table S2). More than 91% of the top fi ve human (344 of cancer maintenance. 375) and >88% of the top fi ve mouse (333 of 375) shRNAs scored >3 (Supplementary Fig. S1C and S1D), a threshold RESULTS score defi ned by positive-control shRNAs. This indicates that the preselection process of 65 shRNAs per gene provided A Functionally Validated RNAi Library Targeting suffi cient coverage to identify several very potent shRNAs KRAS Pathway Genes per gene. To identify potent shRNAs targeting the RAS network, we applied the Sensor assay to evaluate candidate shRNA Sensor siRNAs Are Potent and On-Target sequences targeting 75 human genes and their mouse Synthetic shRNAs and siRNAs enter the endogenous orthologs ( Fig. 1A ). These genes encode many classes of microRNA pathway at different stages, but ultimately use proteins, including kinases, GTPases, and transcription fac- the same conserved machinery to downregulate their target tors. The Sensor assay interrogates large numbers of shRNAs genes. We thus hypothesized that potent shRNA sequences under conditions of single genomic integration (“single- could be directly converted into potent siRNA triggers. On copy”) for their ability to repress a cognate target sequence the basis of top-scoring Sensor shRNA sequences, we gener- placed downstream of a fl uorescent reporter expressed in cis ated corresponding 22mer Sensor siRNAs targeting human (14 ). Although we have previously established this assay and KRAS, KSR1, ARAF, BRAF, RAF1/CRAF, MAP2K1, MAP2K2, shown its potential to identify very potent shRNA sequences MAPK1, MAPK3, PIK3CA, PIK3CB, PIK3CD, AKT1, AKT2, and through full gene tiling, here we applied it to evaluate the effi - AKT3 . We then measured the knockdown effi ciency of three ciency of preselected candidate sequences targeting a much top Sensor shRNAs and two corresponding siRNAs target- larger set of genes to establish a functionally validated RNAi ing endogenous KRAS , and found that Sensor siRNAs retain library. To assemble the initial candidates, 65 shRNAs per >80% mRNA knockdown when transfected at concentrations gene were selected using a combination of bioinformatics as low as 0.5 nmol/L ( Fig. 2A and 2B and Supplementary predictions ( 15 ) and “Sensor rules” requiring shRNA-specifi c Fig. S2A). We extended this validation to ARAF , BRAF , and features
Load more

Recommended publications
  • The Capacity of Long-Term in Vitro Proliferation of Acute Myeloid
    The Capacity of Long-Term in Vitro Proliferation of Acute Myeloid Leukemia Cells Supported Only by Exogenous Cytokines Is Associated with a Patient Subset with Adverse Outcome Annette K. Brenner, Elise Aasebø, Maria Hernandez-Valladares, Frode Selheim, Frode Berven, Ida-Sofie Grønningsæter, Sushma Bartaula-Brevik and Øystein Bruserud Supplementary Material S2 of S31 Table S1. Detailed information about the 68 AML patients included in the study. # of blasts Viability Proliferation Cytokine Viable cells Change in ID Gender Age Etiology FAB Cytogenetics Mutations CD34 Colonies (109/L) (%) 48 h (cpm) secretion (106) 5 weeks phenotype 1 M 42 de novo 241 M2 normal Flt3 pos 31.0 3848 low 0.24 7 yes 2 M 82 MF 12.4 M2 t(9;22) wt pos 81.6 74,686 low 1.43 969 yes 3 F 49 CML/relapse 149 M2 complex n.d. pos 26.2 3472 low 0.08 n.d. no 4 M 33 de novo 62.0 M2 normal wt pos 67.5 6206 low 0.08 6.5 no 5 M 71 relapse 91.0 M4 normal NPM1 pos 63.5 21,331 low 0.17 n.d. yes 6 M 83 de novo 109 M1 n.d. wt pos 19.1 8764 low 1.65 693 no 7 F 77 MDS 26.4 M1 normal wt pos 89.4 53,799 high 3.43 2746 no 8 M 46 de novo 26.9 M1 normal NPM1 n.d. n.d. 3472 low 1.56 n.d. no 9 M 68 MF 50.8 M4 normal D835 pos 69.4 1640 low 0.08 n.d.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • Human RALBP1 Peptide (DAG-P1058) This Product Is for Research Use Only and Is Not Intended for Diagnostic Use
    Human RALBP1 peptide (DAG-P1058) This product is for research use only and is not intended for diagnostic use. PRODUCT INFORMATION Antigen Description RALBP1 plays a role in receptor-mediated endocytosis and is a downstream effector of the small GTP-binding protein RAL (see RALA; MIM 179550). Small G proteins, such as RAL, have GDP- bound inactive and GTP-bound active forms, which shift from the inactive to the active state through the action of RALGDS (MIM 601619), which in turn is activated by RAS (see HRAS; MIM 190020) (summary by Feig, 2003 [PubMed 12888294]).[supplied by OMIM, Nov 2010] Specificity Expressed ubiquitously but at low levels. Shows a strong expression in the erythrocytes. Nature Synthetic Expression System N/A Conjugate Unconjugated Sequence Similarities Contains 1 Rho-GAP domain. Cellular Localization Membrane. Procedure None Format Liquid Preservative None Storage Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles. Information available upon request. ANTIGEN GENE INFORMATION Gene Name RALBP1 ralA binding protein 1 [ Homo sapiens (human) ] Official Symbol RALBP1 Synonyms RALBP1; ralA binding protein 1; RIP1; RLIP1; RLIP76; ralA-binding protein 1; DNP-SG ATPase; ral-interacting protein 1; 76 kDa Ral-interacting protein; dinitrophenyl S-glutathione ATPase; Entrez Gene ID 10928 mRNA Refseq NM_006788.3 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-624-4882 Fax: 1-631-938-8221 1 © Creative Diagnostics All Rights Reserved Protein
    [Show full text]
  • Bioinformatics: a Practical Guide to the Analysis of Genes and Proteins, Second Edition Andreas D
    BIOINFORMATICS A Practical Guide to the Analysis of Genes and Proteins SECOND EDITION Andreas D. Baxevanis Genome Technology Branch National Human Genome Research Institute National Institutes of Health Bethesda, Maryland USA B. F. Francis Ouellette Centre for Molecular Medicine and Therapeutics Children’s and Women’s Health Centre of British Columbia University of British Columbia Vancouver, British Columbia Canada A JOHN WILEY & SONS, INC., PUBLICATION New York • Chichester • Weinheim • Brisbane • Singapore • Toronto BIOINFORMATICS SECOND EDITION METHODS OF BIOCHEMICAL ANALYSIS Volume 43 BIOINFORMATICS A Practical Guide to the Analysis of Genes and Proteins SECOND EDITION Andreas D. Baxevanis Genome Technology Branch National Human Genome Research Institute National Institutes of Health Bethesda, Maryland USA B. F. Francis Ouellette Centre for Molecular Medicine and Therapeutics Children’s and Women’s Health Centre of British Columbia University of British Columbia Vancouver, British Columbia Canada A JOHN WILEY & SONS, INC., PUBLICATION New York • Chichester • Weinheim • Brisbane • Singapore • Toronto Designations used by companies to distinguish their products are often claimed as trademarks. In all instances where John Wiley & Sons, Inc., is aware of a claim, the product names appear in initial capital or ALL CAPITAL LETTERS. Readers, however, should contact the appropriate companies for more complete information regarding trademarks and registration. Copyright ᭧ 2001 by John Wiley & Sons, Inc. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic or mechanical, including uploading, downloading, printing, decompiling, recording or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without the prior written permission of the Publisher.
    [Show full text]
  • RALBP1 Purified Maxpab Rabbit Polyclonal Antibody (D01P)
    RALBP1 purified MaxPab rabbit polyclonal antibody (D01P) Catalog # : H00010928-D01P 規格 : [ 100 ug ] List All Specification Application Image Product Rabbit polyclonal antibody raised against a full-length human RALBP1 Western Blot (Tissue lysate) Description: protein. Immunogen: RALBP1 (NP_006779.1, 1 a.a. ~ 655 a.a) full-length human protein. Sequence: MTECFLPPTSSPSEHRRVEHGSGLTRTPSSEEISPTKFPGLYRTGEPSP PHDILHEPPDVVSDDEKDHGKKKGKFKKKEKRTEGYAAFQEDSSGDEAE enlarge SPSKMKRSKGIHVFKKPSFSKKKEKDFKIKEKPKEEKHKEEKHKEEKHKEK KSKDLTAADVVKQWKEKKKKKKPIQEPEVPQIDVPNLKPIFGIPLADAVER Western Blot (Cell lysate) TMMYDGIRLPAVFRECIDYVEKYGMKCEGIYRVSGIKSKVDELKAAYDRE ESTNLEDYEPNTVASLLKQYLRDLPENLLTKELMPRFEEACGRTTETEKV QEFQRLLKELPECNYLLISWLIVHMDHVIAKELETKMNIQNISIVLSPTVQIS NRVLYVFFTHVQELFGNVVLKQVMKPLRWSNMATMPTLPETQAGIKEEI RRQEFLLNCLHRDLQGGIKDLSKEERLWEVQRILTALKRKLREAKRQEC ETKIAQEIASLSKEDVSKEEMNENEEVINILLAQENEILTEQEELLAMEQFLR RQIASEKEEIERLRAEIAEIQSRQQHGRSETEEYSSESESESEDEEELQIIL EDLQRQNEELEIKNNHLNQAIHEEREAIIELRVQLRLLQMQRAKAEQQAQE enlarge DEEPEWRGGAVQPPRDGVLEPKAAKEQPKAGKEPAKPSPSRDRKETSI Western Blot (Transfected lysate) Host: Rabbit Reactivity: Human, Rat Quality Control Antibody reactive against mammalian transfected lysate. Testing: enlarge Storage Buffer: In 1x PBS, pH 7.4 In situ Proximity Ligation Assay Storage Store at -20°C or lower. Aliquot to avoid repeated freezing and thawing. (Cell) Instruction: MSDS: Download Datasheet: Download enlarge Applications Western Blot (Tissue lysate) RALBP1 MaxPab rabbit polyclonal antibody. Western Blot analysis of RALBP1 expression
    [Show full text]
  • REPS2 Antibody (N-Term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # Ap13131a
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 REPS2 Antibody (N-term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP13131a Specification REPS2 Antibody (N-term) - Product Information Application WB, IHC-P,E Primary Accession Q8NFH8 Other Accession NP_004717.2, NP_001074444.1 Reactivity Human, Mouse Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Calculated MW 71534 Antigen Region 153-182 REPS2 Antibody (N-term) - Additional Information REPS2 Antibody (N-term) (Cat. #AP13131a) Gene ID 9185 western blot analysis in mouse brain tissue lysates (35ug/lane).This demonstrates the Other Names REPS2 antibody detected the REPS2 protein RalBP1-associated Eps domain-containing (arrow). protein 2, Partner of RalBP1, RalBP1-interacting protein 2, REPS2, POB1 Target/Specificity This REPS2 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 153-182 amino acids from the N-terminal region of human REPS2. Dilution WB~~1:1000 IHC-P~~1:10~50 Format Purified polyclonal antibody supplied in PBS with 0.09% (W/V) sodium azide. This antibody is purified through a protein A column, followed by peptide affinity purification. REPS2 Antibody (N-term) (Cat. #AP13131a)immunohistochemistry analysis Storage in formalin fixed and paraffin embedded Maintain refrigerated at 2-8°C for up to 2 human cerebellum tissue followed by weeks. For long term storage store at -20°C peroxidase conjugation of the secondary in small aliquots to prevent freeze-thaw antibody and DAB staining.This data cycles. demonstrates the use of REPS2 Antibody (N-term) for immunohistochemistry. Clinical Precautions relevance has not been evaluated.
    [Show full text]
  • Supplementary Table 1: Genes Located on Chromosome 18P11-18Q23, an Area Significantly Linked to TMPRSS2-ERG Fusion
    Supplementary Table 1: Genes located on Chromosome 18p11-18q23, an area significantly linked to TMPRSS2-ERG fusion Symbol Cytoband Description LOC260334 18p11 HSA18p11 beta-tubulin 4Q pseudogene IL9RP4 18p11.3 interleukin 9 receptor pseudogene 4 LOC100132166 18p11.32 hypothetical LOC100132166 similar to Rho-associated protein kinase 1 (Rho- associated, coiled-coil-containing protein kinase 1) (p160 LOC727758 18p11.32 ROCK-1) (p160ROCK) (NY-REN-35 antigen) ubiquitin specific peptidase 14 (tRNA-guanine USP14 18p11.32 transglycosylase) THOC1 18p11.32 THO complex 1 COLEC12 18pter-p11.3 collectin sub-family member 12 CETN1 18p11.32 centrin, EF-hand protein, 1 CLUL1 18p11.32 clusterin-like 1 (retinal) C18orf56 18p11.32 chromosome 18 open reading frame 56 TYMS 18p11.32 thymidylate synthetase ENOSF1 18p11.32 enolase superfamily member 1 YES1 18p11.31-p11.21 v-yes-1 Yamaguchi sarcoma viral oncogene homolog 1 LOC645053 18p11.32 similar to BolA-like protein 2 isoform a similar to 26S proteasome non-ATPase regulatory LOC441806 18p11.32 subunit 8 (26S proteasome regulatory subunit S14) (p31) ADCYAP1 18p11 adenylate cyclase activating polypeptide 1 (pituitary) LOC100130247 18p11.32 similar to cytochrome c oxidase subunit VIc LOC100129774 18p11.32 hypothetical LOC100129774 LOC100128360 18p11.32 hypothetical LOC100128360 METTL4 18p11.32 methyltransferase like 4 LOC100128926 18p11.32 hypothetical LOC100128926 NDC80 homolog, kinetochore complex component (S. NDC80 18p11.32 cerevisiae) LOC100130608 18p11.32 hypothetical LOC100130608 structural maintenance
    [Show full text]
  • Quantitative Trait Loci Mapping of Macrophage Atherogenic Phenotypes
    QUANTITATIVE TRAIT LOCI MAPPING OF MACROPHAGE ATHEROGENIC PHENOTYPES BRIAN RITCHEY Bachelor of Science Biochemistry John Carroll University May 2009 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY December 2017 We hereby approve this thesis/dissertation for Brian Ritchey Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies by ______________________________ Date: _________ Dissertation Chairperson, Johnathan D. Smith, PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, David J. Anderson, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Baochuan Guo, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Stanley L. Hazen, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Renliang Zhang, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Aimin Zhou, PhD Department of Chemistry, Cleveland State University Date of Defense: October 23, 2017 DEDICATION I dedicate this work to my entire family. In particular, my brother Greg Ritchey, and most especially my father Dr. Michael Ritchey, without whose support none of this work would be possible. I am forever grateful to you for your devotion to me and our family. You are an eternal inspiration that will fuel me for the remainder of my life. I am extraordinarily lucky to have grown up in the family I did, which I will never forget.
    [Show full text]
  • Dysregulation of Micro-143-3P and BALBP1 Contributes to the Pathogenesis of the Development of Ovarian Carcinoma
    ONCOLOGY REPORTS 36: 3605-3610, 2016 Dysregulation of micro-143-3p and BALBP1 contributes to the pathogenesis of the development of ovarian carcinoma Hongyan ZHANG1 and WANBIN LI2 1Department of Gynecology, Affiliated Hospital of Jining Medical University, Jining, Shandong 272029; 2Jining Medical University, Jining, Shandong 272113, P.R. China Received February 28, 2016; Accepted April 9, 2016 DOI: 10.3892/or.2016.5148 Abstract. The objective of the present study was to identify the inhibitor. These findings provide support that downregula- association between mir-143-3p and RalA-binding protein 1 tion of the miR-143-3p is associated with a decreased risk of (RALBP1), and their roles in regulating the development of ovarian cancer. ovarian cancer. Overexpression of RALBP1 induced apoptosis of the ovarian cancer cells, and developed ovarian cancer. Introduction In silico analysis and luciferase assay were used to identify whether RALBP1 was the target of mir-143-3p. Subsequently, Ovarian cancer is the most life-threatening malignancy in real-time PCR and western blotting were used to determine reproductive tract and fourth leading cause of cancer-related the expression level of mir-143-3p, RALBP1 mRNA and deaths in women. Majority of ovarian cancers stem from protein in different groups, furthermore, MTT assay and flow epithelium, though there are also stromal and germ cell cytometry were used to detect the viability and apoptosis of tumors (1,2). Generally, ovarian tumors are silent at early stage cells in different treatment groups. We identified RALBP1 and remain unrecognized in most cases (>80%) until ovarian as a target gene of miR-143-3p using computational analysis, carcinoma has metastasized to other areas out of the ovaries.
    [Show full text]
  • Rlip Depletion Prevents Spontaneous Neoplasia in TP53 Null Mice
    Rlip depletion prevents spontaneous neoplasia in TP53 null mice Sanjay Awasthia,b,1, Joshua Tompkinsb, Jyotsana Singhalb, Arthur D. Riggsb,1, Sushma Yadavb, Xiwei Wuc, Sharda Singha, Charles Wardenc, Zheng Liud, Jinhui Wangc, Thomas P. Slavine, Jeffrey N. Weitzele, Yate-Ching Yuand, Meenakshi Awasthib, Satish K. Srivastavaf, Yogesh C. Awasthif, and Sharad S. Singhalb aDivision of Hematology & Oncology, Department of Internal Medicine, Texas Tech Health Sciences Center, Lubbock, TX 79430-9410; bDiabetes and Metabolism Research Institute, City of Hope Comprehensive Cancer Center, Duarte, CA 91010; cIntegrative Genomics Core, City of Hope Comprehensive Cancer Center, Duarte, CA 91010; dBioinformatics Core, City of Hope Comprehensive Cancer Center, Duarte, CA 91010; eDivision of Clinical Cancer Genomics, City of Hope Comprehensive Cancer Center, Duarte, CA 91010; and fDepartment of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555-0647 Contributed by Arthur D. Riggs, February 7, 2018 (sent for review November 9, 2017; reviewed by Shivendra Singh and Dan Theodorescu) TP53 (p53) is a tumor suppressor whose functions are lost or electrophilic toxins (9–30). Because Rlip-catalyzed efflux of GS-E altered in most malignancies. p53 homozygous knockout (p53−/−) prevents product/feedback inhibition of several mercapturic acid mice uniformly die of spontaneous malignancy, typically T-cell pathway enzymes, its loss promotes apoptosis exerted by xenobiotics lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic and GS-E, derived from oxidative degradation of ω-6 fatty acids acid pathway transporter protein that also functions as a Ral effec- (31). Its ATPase activity is coupled with clathrin-dependent endo- tor involved in clathrin-dependent endocytosis.
    [Show full text]
  • RLIP76: a Structural and Functional Triumvirate
    cancers Review RLIP76: A Structural and Functional Triumvirate Jasmine Cornish, Darerca Owen and Helen R. Mott * Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK; [email protected] (J.C.); [email protected] (D.O.) * Correspondence: [email protected] Simple Summary: The RLIP76 protein is present at high levels in multiple cancers, compared with levels in normal cells. In cancer cells it is thought to be important for removal of chemotherapeutics, while in normal cells it has been implicated in endocytosis, stress response and mitochondrial fission. Although RLIP76 is a potential target for ovary, breast, lung, colon, prostate and kidney cancers, only the middle third of the protein has been structurally characterized. Understanding the full structure of RLIP76 will help us to better understand the signalling pathways in which it is involved and by extension its role in cancer. Abstract: RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may Citation: Cornish, J.; Owen, D.; Mott, mediate membrane association, consistent with its role in transport.
    [Show full text]